Biosensors are analytical tools that detect the presence of a chemical or biochemical species in a complex mixture by combining the molecular recognition properties of biological macromolecules (e.g., enzymes, antibodies, DNA or even whole cells) with signal transduction mechanisms (e.g., optical or electrochemical) that couple ligand bindings with readily detectable physical changes.
The optical signal transduction mechanisms employed by biosensors are based on absorption spectroscopy (ultraviolet (UV) to deep infrared (IR)), Raman or fluorescence spectroscopy. Fluorescence spectroscopy provides a particularly important detection mechanism because a number of biological species (including the green fluorescent protein (GFP), nucleic acids and flavine nucleotides) are naturally fluorescent. In addition, biological species that are not naturally fluorescent may be chemically bound to fluorescent molecules (known as labels).
To increase analytical throughput, an analytical process should be capable of simultaneously detecting a number of different species. Biosensor arrays achieve this by assembling a large number of different biological macromolecules (each of which contains a recognition site for a given biological species, with such species being known henceforth as an analyte) into densely packed arrays of unique sensor elements.
Biosensor arrays have three main operational mechanisms, namely labeled analyte pooling (used in DNA and RNA hybridization assays), sandwich assays (used for antibody recognition) and direct assays.
Referring to FIG. 1, during labeled analyte pooling a biological macromolecule 10 specific for an analyte 12 is immobilized on a solid support 14. A sample (containing an analyte 12) is mixed with a solution of a fluorescent label 16 that binds to the analyte 12 therein. The sample is then introduced to the biosensor array and the analyte 12 therein is bound to the biological macromolecule 10 specific therefore. Other species 18, 20 in the sample that are not of analytical interest (and for which there are no biological macromolecules immobilized on the solid support 14) remain in free solution.
The support 14 is then washed with a cleaning solution (not shown) and any unbound species in the sample are flushed therefrom, leaving the fluorescently labeled analyte 12 bound to the support 14. The fluorescently labeled analyte 12 fluoresces when exposed to radiation (e.g., from an IR laser) and the resulting fluorescent pattern of the biosensor array acts as a biochemical fingerprint that can be readily imaged.
Referring to FIG. 2, a fluorescence biosensor 30 typically comprises a stimulating light source 32, a substrate 34 and a photodetector 36. The substrate 34 comprises a plurality of sensor elements 37a, 37b and 37c each of which comprises an immobilized biological macromolecule 38a, 38b and 38c specific for a particular analyte of interest. While FIG. 2 shows the photodetector 36 disposed remotely from the substrate 34, nonetheless, it will be appreciated that this arrangement is not essential and the substrate 34 could alternatively be configured to house both the photodetector 36 and the sensor elements 37a, 37b and 37c. 
Using, for example, the above-described labeled analyte pooling scheme, analytes 40a and 40c in a sample have fluorescent labels bound thereto. When the sample (not shown) is introduced to the substrate 34, the labeled analytes 40a and 40c bind to the appropriate macromolecule 38a and 38c. However, if an analyte that binds to a particular macromolecule 38b is not present in the sample, the corresponding sensor element 37b remains free of labeling.
The light source 32 emits light 42 of wavelength λ1, which is a stimulating wavelength for the fluorescent labels (bound to the analytes 40a and 40c). The light source 32 is positioned so that the light 42 it emits falls upon the sensor elements 37a, 37b and 37c (and any fluorescently labeled analytes bound thereto). It will be appreciated that there may be some additional optical elements (e.g., lens, lightguide, etc.) disposed between the light source 32 and the sensor elements 37a, 37b and 37c. It will also be appreciated that the light source 32 may alternatively scan the array of sensor elements 37a, 37b and 37c. The light 42 stimulates the fluorescent labels bound to the analytes 40a and 40c to emit radiation of wavelength λ2 (λ1<λ2)
The photodetector 36 comprises a plurality of pixels 44a, 44b and 44c, each of which is positioned to detect the radiation emitted from a given sensor element 37a, 37b and 37c. As before, it will be appreciated that there may be some additional optical elements (e.g. lens, a light guide, etc.) disposed between the sensor elements 37a, 37b and 37c and the photodetector 36. It will also be appreciated that the photodetector 36 may alternatively scan the array of sensor elements 37a, 37b and 37c. 
While the biochemistry of immobilizing biological macromolecules, etc. is relatively well established, one of the main problems which remains to be addressed arises because the radiation λ2 emitted from the fluorescently labeled analytes 40a and 40c is very weak compared to the radiation 42 emitted by the light source 32.
There are a number of devices currently on the market for analyzing biological samples. For example, the Agilent 2100 bioanalyzer uses microfluidic technology to enable electrophoretic separation of biological components (detected by fluorescence) and flow cytometric analysis of cell fluorescence parameters. The Agilent 2100 bioanalyzer uses a laser (as a stimulating light source) to scan the relevant biological components and a photomultiplier tube to detect the weak fluorescence signal emitted therefrom.
Unfortunately, prior art systems such as the Agilent 2100 bioanalyzer are typically large, very expensive and not amenable to miniaturization. In addition, the process of optimizing the focus of the stimulating light source (to compensate for tilting of the specimen and/or non-flat surfaces) can be quite complicated.
Finally, since the Agilent 2100 bioanalyzer scans each sensor element (e.g., DNA binding site) serially, the scanning time of the bioanalyzer is dependent on the number of sensor elements on a substrate. Consequently, if a substrate contains a number of sensor elements (to detect multiple different analytes) the scanning time of the Agilent 2100 bioanalyzer may be quite long.
At present, fluorescent biosensor typically use charge coupled devices (CCDs) or complementary metal oxide (CMOS) cameras as photodetectors. Sometimes dichroic mirrors are used to separate the radiation from the stimulating light source (known as stimulating radiation) from the radiation emitted by the fluorescent labels (known as fluorescent radiation) on the basis of the difference in their wavelengths (i.e., λstim<λfluor). However, these wavelength discrimination systems are typically large and expensive.
One way of avoiding the problem of the large size of such wavelength discrimination systems is to perform temporal discrimination by synchronizing the operation of the biosensor's stimulating light source and photodetector. To illustrate this point, FIG. 3 shows the relative timing of:
(a) an activation signal (STIM) for a biosensor's stimulating light source;
(b) a reset signal (RESET) of the biosensor's CCD/CMOS detector; and
(c) a voltage (Vpd) generated in the biosensor's CCD/CMOS detector.
Referring to traces (a) and (b) in FIG. 3, it can be seen that the stimulating light source is activated in a pulsating fashion, which is timed to match the timing of the reset signal. The CCD/CMOS detector is reset by a pulse 50 while the fluorescent labels are being stimulated by a pulse 52 from the stimulating light source (trace (a)). Thus, the CCD/CMOS does not start to measure radiation until after the stimulating light source pulse 52 (trace (a)) is ended and the only light present is that emitted from the fluorescent labels.
However, as the amount of light emitted by a fluorescent label is quite small, a comparatively small voltage (67V) is generated thereby in the CCD/CMOS detector during measurement time interval (τ). Thus, the sensitivity of a CCD/CMOS detector to such fluorescent radiation becomes particularly important.
One way of increasing the sensitivity of a CCD/CMOS detector would be to increase the size of its pixels. As biosensor arrays comprise a small number of sensor elements compared with the number of pixels in a conventional CCD/CMOS detector, such CCD/CMOS detectors possess more than enough resolution to image the biosensor array. Consequently, there is no practical impediment to scanning biosensor arrays with such larger pixels, wherein a charge integrator circuit as depicted in FIG. 4 is particularly suitable for processing the signals acquired by such enlarged pixels.
Referring to FIG. 4, the charge integrator circuit 54 comprises a charge amplifier 56 connected in parallel with a feedback capacitor Cfb and a reset switch 58. The charge integrator circuit 54 is in turn connected to a comparator 60 which permits the conversion of the analog output from the charge integrator circuit 54 into a digital value by ramping a reference signal (Vref) (from a digital to analog converter (DAC), for example) and using the comparator's 60 output to store the resulting digital value.
The output from the charge integrator circuit 54 may also be converted to a digital signal by connecting an analog to digital converter (ADC) to the charge integrator circuit 54 (instead of the comparator 60) Alternatively, the configuration shown in FIG. 4 (with the comparator 60 connected to the output of the charge integrator circuit 54) may be used with the reference signal (Vref) held constant and measuring the amount of time for the output from the charge integrator circuit 54 to reach the level of the reference signal (i.e. , until Vout=Vref).